tbuckley/cabot-search huggingface dataset:
Abstract P3175: Mitochondrial Fission And Extracellular Mitochondria Mediate Cardiac Dysfunction In Obesity Induced Cardiomyopathy
Authors: Sin-Jin Li, Ching-Yi Chen, Nicolai Ostberg, Laura Tetri, Ivan Lopez, Timothy Cornell, Daria Mochly Rosen, Bereketeab Haileselassie
Journal: Circulation Research (2023)

Background: Recent studies have demonstrated that dysregulation of mitochondrial dynamics, including excessive mitochondrial fission, lead to extracellular release of damaged mitochondria (exMito) as an alternate mechanism for mitochondrial quality control. However, the exact role of this mechanism on obesity cardiomyopathy (OCM) has not been explored. This project aims to determine if Drp1/Fis1 dependent excessive mitochondrial fission mediates the release of damaged mitochondria and propagate cardiomyocyte injury in OCM. Methods: H9c2 cardiomyoblast were treated with 200 μM palmitate (PA) to induce lipotoxicity. C57BL6/J mice were subjected to a high-fat diet (HFD) for 12 weeks to induce OCM. Changes in cardiac Drp1 activity, mitochondrial morphology, bioenergetics and contractile function was evaluated. Simultaneously, exMito were collected and characterized from supernatant and plasma. Using peptide P110 co-treatment , we determined the impact of pharmacologic inhibition of Drp1/Fis1 dependent mitochondrial fission on cardiac mitochondrial function as well as the quality and quantity of exMito. Results: H9c2 cells treated with PA demonstrated excessive mitochondrial fission, increased oxidative stress and decreased ATP level. The excessive mitochondrial fission seen following PA treatment was associated with the release of damaged exMito. The exMito isolated from PA treated cells were noted to mediate cytotoxicity to healthy H9c2s independent of PA. P110 treatment was protective against PA mediated mitochondrial damage, represented by improved bioenergetic profile, increased ATP and lower oxidative stress. P110 treatment also lowered the release of damaged mitochondria from PA treated H9c2s. Similarly, treatment of HFD mice with P110, improved mitochondrial function and glucose utilization, exMito release and subsequently improved cardiomyocyte contractile function. Conclusion: Lipotoxicity induced mitochondrial fission, disrupted mitochondrial function leading to release of exMito. P110 limited lipotoxicity mediated alterations in mitochondrial dynamics, mitochondrial function and exMito, and then improved cardiac function. Therefore, pathologic fission and exMito may play a critical role in OCM.
Abstract P3181: A Cyclophilin D-mitochondria-redox Differentiation Pathway Regulates Neonatal Heart Bioenergetics And Function
Authors: Hope Kile, Joshua Agyarko, Gisela Beutner, George A Porter
Journal: Circulation Research (2023)

Background: During birth, demands on the heart increase when exposed to high oxidative stress. Cardiac myocyte differentiation is dependent on changes in mitochondrial function that regulate cell stress pathways to enhance differentiation. We have shown these changes are dependent on closure of the mitochondrial permeability transition pore (mPTP) controlled by the protein cyclophilin D (CypD). These studies aim to determine the effects of independently manipulating CypD, mitochondrial membrane potential, and reactive oxygen species (ROS) production on mitochondrial function, myocyte differentiation, and cardiac function in the neonate. Methods: Neonatal C57Bl6 mice were intraperitoneally injected on postnatal day (P) 1-6 of with mitoparaquat (0.025-0.05 mg/kg), Tempol (0.7 mg/kg), mitoTempo (0.7 mg/kg), 2,4-dinitrophenol (2 mg/kg), or vehicle. Hearts were harvested at P7 and mitochondrial respiration, enzymatic activity of the electron transport chain (ETC) complex (Cx) I and 2, and assembly of mitochondrial supercomplexes were measured by native gel electrophoresis. Results: Treatment with mitoparaquat to increase mitochondrial ROS caused a dose-dependent decrease of Cx I activity measured by oxygen consumption and enzyme assays. This corresponded to decrease in assembly of Cx-I containing supercomplexes. Decreasing mitochondrial ROS using mitoTempo had no effect on Cx I activity but increased assembly of Cx-III containing supercomplexes. Decreasing ROS in the cell with Tempol decreased Cx-I activity but had no effect on supercomplex assembly. Uncoupling at the mitochondrial membrane using 2,4-dinitrophenol increased electron flux from Cx I to Cx III but may decrease supercomplex assembly. Treatment of isolated mitochondria with cyclosporin A to inhibit CypD had no effects on these measures. Conclusions: Changes in ROS and mitochondrial membrane potential have selective effects on ETC assembly, activity in the neonatal period, and are not inhibited by closure of the mPTP. This data suggests a pathway where CypD regulates the mPTP to regulate the ETC, membrane potential, and ROS production to regulate myocyte differentiation; such a pathway could be targeted to increase cardiac maturation in infants with poor cardiac function.
Abstract EC.04: The Effects Of Cardiac Troponin Activator CK-358 On Cardiac Contractility In A Mouse Model Of Genetic Dilated Cardiomyopathy
Authors: Stephanie Neys, Kristina Kooiker, Amy Martinson, Darren Hwee, James Hartman, Anne N Murphy, Bradley Morgan, Jennifer Davis, Fady Malik, Farid Moussavi-Harami
Journal: Circulation Research (2023)

Genetic dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. However, most treatments do not address the underlying contractile deficit caused by the disease. In an effort to create more targeted DCM therapeutics, novel small molecule drugs known as sarcomeric modulators have been of recent interest. In a recent study, a cardiac troponin activator was shown to improve cardiac muscle contractility without adversely affecting energetics or diastolic function. Here, we aim to elucidate the mechanism by which the cardiac troponin activator CK-3827358 (CK-358) is able to recover contractile function in a mouse model of DCM with a mutation at residue 61 of cardiac troponin C (cTnC I61Q). We measured steady state force and k tr , a measure of crossbridge cycling kinetics, in demembranated ventricular tissue from control and cTnC I61Q mice in solutions with increasing concentrations of calcium, ranging from pCa 9 to pCa 4. The cTnC I61Q mutation significantly decreased calcium sensitivity versus control (pCa 50 : 5.48 ± 0.06 vs 5.75 ± 0.01). Compared to controls, CK-358 (1 μM) significantly increased calcium sensitivity (pCa 50 ) in tissue from both cTnC I61Q and control mice (6.03 ± 0.06 and 6.50 ± 0.18, P=0.005) without any change in maximum force. CK-358 also significantly increased k tr , possibly suggesting activation of thin filaments. Next, we used a fluorescently labeled human cardiac troponin C (HcTnC) conjugated to N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) combined with subunits T and I to make a full HcTn complex. We performed calcium titrations with the HcTn-IANBD and saw no change in fluorescence in the control protein with and without CK-358. Finally, we performed HcTnC titrations with HcTnI and saw no change in fluorescence at maximum, sub-maximum, and minimum calcium concentrations. Overall, we demonstrate that cardiac troponin activator CK-358 did not alter calcium binding to HcTnC or its interaction with HcTnI. However, it restores the calcium sensitivity of cTnC I61Q DCM ventricular sarcomeres. Future studies will examine the effects of CK-358 on calcium binding in decorated thin filaments and measure activation using stopped flow spectroscopy in this model of DCM.
Abstract P3070: Aortic Valve Calcification Is Accelerated By DNMT3A And TET2 Gene Driver Mutations Associated With Clonal Hematopoiesis
Authors: Wesley T Abplanalp, Bianca Schuhmacher, Silvia Mas-Peiro, Maria A Zuriaga, Nuria Matesanz, Jose J Fuster, David John, Marion Muhly-Reinholz, Mariuca Vasa-Nicotera, Maximilian Merten, Stefanie Dimmeler, Andreas M Zeiher
Journal: Circulation Research (2023)

Background: Clonal hematopoiesis (CH) due to DNMT3A or TET2-driver mutations associates with heightened inflammation and a worse prognosis among patients with severe calcific aortic valve stenosis (AS). However, it is unknown what role CH plays in the pathogenesis of AS. AS is the most common age-related heart valve disease, with no medical therapy to halt progression. Methods and Results: Single-cell RNA-sequencing of immune cells of CAVD patients (n=13) with and without the most prevalent CH-mutations (DNMT3A and TET2), having a mean CH-driver gene variant allele frequency was 6.5% for DNMT3A and 17.9% for TET2. Monocytes of patients harboring DNMT3A and TET2 mutations shared 836 upregulated genes, which were associated with glycolytic, pro-inflammatory activation (CXCL10, IL38) along with paracrine pro-calcific factors (Oncostatin M (OSM), S100A9), signifying that CH might be causally involved in AS. Indeed, silencing of TET2 or DNMT3A in macrophages promotes M1-like polarization and increases release of pro-calcific mediators, particularly S100A9 and OSM. To assess whether CH-gene silenced macrophages may stimulate mesenchymal cells to secrete calcium, secreted factors from TET2 or DNMT3A-silenced macrophages were applied mesenchymal cells. Indeed, CH-gene silenced supernatants induced osteoblastic differentiation of mesenchymal cells, evidenced by increased Alizarin red calcium staining and elevated levels of ALP and RUNX2. This CH-mediated enhancement in osteoblastic transformation could be ablated by silencing of OSM. Finally, valves from atheroprone Ldlr-/- mice receiving Tet2-/- bone marrow transplants demonstrated increased total calcified area (p&lt;0.05, 1.49-fold) along with increased numbers of calcification deposits (p&lt;0.05, 2.33-fold) as shown by von Kossa staining, along with enhanced OSM and S100A9 levels. Conclusion: This is the first study to show that somatic CH-driver mutations in monocytes may accelerate AS. Moreover, calcification was ablated via silencing of OSM in CH-gene silenced macrophages. These data suggest Oncostatin M may be a therapeutically promising target in halting the development of AS in patients harboring CH mutations.
Abstract P3201: Fatty Acid Oxidation Decreases Independently Of Up-regulated Glucose Utilization In The Setting Of Elevated Afterload
Authors: Hande C Piristine, Herman I May, Nan Jiang, Francisco Olivares-Silva, Abdallah Elnwasany, Pamela Szweda, Luke I Szweda, Caroline Kinter, Michael T Kinter, Gaurav Sharma, Xiaodong Wen, Craig Malloy, Michael E Jessen, Thomas G Gillette, Joseph A Hill
Journal: Circulation Research (2023)

Background: To support the unremitting contractile function of the heart, free fatty acids (FFA) are the substrate of choice, with a shift toward greater reliance on glucose when increased cardiac output is required. However, in the setting of obesity or diabetes, this metabolic flexibility is impaired. The putative necessity of a shift in substrate utilization has been hypothesized to occur due to the lower oxygen requirement of glucose versus fat metabolism, but this has never been tested conclusively. Furthermore, in the case of increased afterload, whereas oxygen demand increases, myocardial oxygen availability remains adequate for fatty acid oxidation (FAO). Therefore, a “preference” for glucose has been proposed. Methods and Results: Pyruvate dehydrogenase complex (PDC) is the rate-limiting enzyme linking glycolysis to the TCA cycle. By over-expressing PDC kinase 4 (PDK4) in cardiomyocytes, we ensured that PDC was phosphorylated and thereby inhibited. This leads to diminished use of pyruvate, limiting energy production primarily to FAO. Unlike high fat diet feeding, which also increases PDK4 and inhibits PDH, this model positioned us to query the impact of PDH inhibition under elevated workload demand in the absence of systemic high fat conditions. As expected, PDK4 transgenic mice manifested normal cardiac output at baseline. However, they manifested a rapid and severe decline in contractile function when challenged with increased afterload triggered by either L-NAME or surgical transverse aortic constriction (TAC). Metabolic flux analysis revealed that, after TAC, fractional FAO decreased - surprisingly - independent of an increase in glucose/pyruvate utilization. Additionally, proteins involved in the transport and oxidation of FFA were paradoxically down-regulated after TAC regardless of genotype. Conclusions: These data demonstrate that cardiomyocytes in a setting in which glucose utilization is clamped and prevented from increasing do not compensate for the deficit in glucose utilization by up-regulating FFA use. In aggregate, our findings suggest that reliance on glucose in the setting of pathological cardiac stress is largely a necessity rather than a simple preference.
Abstract P3093: Early Myocarditis In The Desmoglein 2 Mutant Mouse Model Of Arrhythmogenic Cardiomyopathy
Authors: Steven Valdez, Lindsey Taylor, Rachel Rosenzweig, Shyam S Bansal, Joseph Palatinus
Journal: Circulation Research (2023)

Arrhythmogenic cardiomyopathy (ACM) is an inherited disease caused by mutations in desmosome proteins. Patients with ACM are born with normal hearts but develop arrhythmias, fibrofatty infiltration, and sudden death. The desmoglein 2 (DSG2) mutant mouse is a well-established model of ACM that recapitulates the human phenotype of the disease. This mouse develops severe ventricular arrhythmias, systolic dysfunction, myocardial fibrosis and exercise induced sudden death. Here, we present evidence of early myocardial immune cell infiltration as well as an inverse relationship between cardiac function and adult myocardial cytokine expression in DSG2 mutant mice. We examined 20 week old adult DSG2 homozygote mice and wildtype littermate controls for systolic function using echocardiography, cytokine expression using rtPCR and fibrosis using masons trichrome staining. Consistent with previous studies we found ventricular fibrosis is significantly increased in mutant hearts (p=0.003). We demonstrate that osteopontin, an inflammatory cytokine released by macrophages, is highly expressed in these hearts (p=2.0x10 -4 ). We detected an inverse correlation between systolic function and the osteopontin expression levels in these mutant mice (p=0.0015, r=-0.8315, R 2 =0.6914). These results lead us to suspect that immune cell infiltration may contribute to the disease progression. Subsequently, we examined immune cell populations in 3 week old DSG2 hearts, a period of time before the development of fibrosis, systolic dysfunction or arrhythmias. We measured &gt;40-fold higher number of CD45 + leukocytes (with increased Ly6G + neutrophils and Ly6C + monocytes) within the heart muscle of DSG2 homozygotes via flow cytometry (p=7.4x10 -5 ), consistent with myocarditis. These results suggest myocarditis precedes fibrosis in the DSG2 mutant mouse model. Our ongoing work is focused on further characterizing the immune response in early post-natal life in the DSG2 mouse heart and determining whether early myocarditis contributes to the phenotype in ACM. Our results highlight potentially novel therapeutic targets to prevent disease progression in ACM.
Abstract GS.06: The Fibrogenic Transcription Factor Sox9 Controls Mesenchymal Activation Of Endothelial Cells And Drives Fibrotic Disease
Authors: Felix A Trogisch, Aya Abouissa, Merve Keles, Anne Birke, Manuela Fuhrmann, Gesine M Dittrich, Julio Cordero, Adel El Sherbiny, Abel Martin Garrido, Steve Grein, Luka Nicin, Sophia-Iris Bibli, Rannar Airik, Andreas Kispert, Ralf Kist, Sun Quanchao, Sina W Kürschner, Manuel Winkler, Norbert Gretz, Carolin Mogler, Thomas Korff, Philipp-Sebastian Koch, Stefanie Dimmeler, Gergana Dobreva, Joerg Heineke
Journal: Circulation Research (2023)

Introduction: Endothelial cells (ECs) undergo mesenchymal activation (EndoMA) during the onset of fibrotic cardiac remodeling. The contribution of EndoMA to disease progression, matrix deposition and upstream master regulators, however, remain elusive. Objective: Aim of the study was to investigate the impact of the fibrogenic transcription factor Sox9 in ECs on the development of fibrotic disease. Methods &amp; Results: Following both transverse aortic contriction (TAC) and administration of high fat diet (HFD) with NOS inhibitor L-NAME in mice, as well as in human diseased hearts, we found a significant induction of Sox9 in cardiac ECs. EC-specific overexpression of Sox9 in transgenic mice induced fibrotic and hypertrophic remodeling of heart, lung, liver and spleen, accompanied by left-ventricular diastolic followed by systolic dysfunction. Both bulk and single-cell (SC) RNA sequencing (seq) of isolated ECs as well as immunofluorescence staining showed that ECs were the origin of ECM secretion. In turn, EC-specific Sox9 inactivation in mice protected from heart failure following TAC or HFD + L-NAME, resp., as indicated by preserved cardiac function, reduced cardiac hypertrophy and fibrosis. In-depth analysis of both bulk and SC RNAseq of isolated cardiac ECs revealed that Sox9 regulates mesenchymal, inflammatory and cell-to-cell communication-associated gene ontologies. Interestingly, Sox9 expression was limited to a specific EC cluster following TAC, in which Sox9 and ECM genes were co-expressed. Endothelial Sox9 inactivation, markedly reduced ECM gene expression in ECs from this cluster, including Ccn2 , which encodes for CTGF. Both ATAC and CUT&amp;TAGseq of transgenic mice suggested direct induction of Ccn2 by Sox9 in cardiac ECs. Interestingly, fibroblasts (FBs) co-cultivated with SOX9-overexpressing HUVECs displayed increased levels of mesenchymal gene expression and enhanced migration. This effect was mitigated by knock-down of CCN2 , suggesting that SOX9 triggers CTGF-mediated signaling from ECs leading to paracrine pro-fibrotic activation of FBs. In conclusion, ECs are critical drivers of fibrotic cardiac remodeling by upregulating Sox9, which triggers EndoMA in ECs, leading to ECM secretion from ECs and paracrine FB activation.
Abstract P1149: Junctophilin-2 Promotes Cardiomyocyte Survival By Blocking Murf1-mediated Junctin Ubiquitination And Proteasome-dependent Degradation
Authors: Xiaoyun Ji, Yifan Huang, Rui Ni, Guo-Chang Fan, Dong Zheng, Douglas L Jones, Long-Sheng Song, Subrata Chakrabarti, Zhaoliang Su, Tianqing Peng
Journal: Circulation Research (2023)

Background: Junctophilin-2 is required for the development, maturation and integrity of the t-tubule system and the gating stability of RyR2 in cardiomyocytes. This study investigated whether and how junctophilin-2 maintained junctin, a scaffold protein stabilizing RyR2, to prevent cardiomyocyte death under stress. Methods: Cardiomyocytes were exposed to conditions of stress including palmitate, doxorubicin, or hypoxia/re-oxygenation. Adenoviral vectors were employed to manipulate expression of junctophilin-2 and junctin in cardiomyocytes. Molecular/cellular/biochemical analyses were conducted. Results: Different conditions of stress decreased junctophilin-2 expression through aberrant autophagy and concomitantly induced a reduction of junctin protein in cardiomyocytes. Over-expression of junctophilin-2 preserved the protein levels of junctin and attenuated cytosolic Ca 2+ and apoptosis in cardiomyocytes under stress. Knockdown of junctophilin-2 reproduced the detrimental phenotypes of stress in cardiomyocytes. Notably, over-expression of junctin prevented cardiomyocyte death under stress whereas knockdown of junctin offset the protective effects conferred by junctophilin-2 over-expression. Mechanistically, junctophilin-2 blocked MURF1-junctin interaction thereby preventing junctin ubiquitination and proteasome-dependent degradation. Mass spectrometry analysis identified multiple ubiquitination sites on the junctin protein and the non-ubiquitinated junctin mutant (K8R/K102R/K107R/K140R) was resistant to degradation. Conclusions: This study uncovers an unrecognized role of junctophilin-2 in preventing junctin ubiquitination and degradation in maintaining cytosolic Ca 2+ homeostasis. Junctophilin-2 and junctin represent two new survival factors of cardiomyocytes and thus, may be new therapeutic targets for cardiac protection.
Abstract P3041: Stage-specific Alternative Rna Splicing Attunes Cardiomyocyte Proliferation And Contraction
Authors: Peiheng Gan, Zhaoning Wang, Rhonda S Bassel-Duby, Ning Liu, Eric N Olson
Journal: Circulation Research (2023)

During heart development, cardiomyocytes display reduced proliferation and enhanced contractility to support cardiac functional maturation, but the mechanistic basis is not fully understood. Alternative RNA splicing is an important mechanism of RNA posttranscriptional modification and gene regulation in the heart. In studying a cardiac RNA binding protein, RBPMS (RNA-binding protein with multiple splicing), we found that RBPMS mediates distinct RNA splicing profiles in adult hearts versus neonatal hearts, indicating its stage-specific modulation of alternative RNA splicing. We showed that RBPMS regulates the splicing of cytoskeletal genes to enable proliferation of neonatal cardiomyocytes. In adult hearts, RBPMS regulates the splicing of sarcomere genes to support cardiac contraction. Cardiac-specific loss of RBPMS causes severe dilated cardiomyopathy and early lethality in adult mice. Using both in vivo and in vitro approaches, we found that the absence of RBPMS caused aberrant splicing of some key sarcomeric components, including Titin and Nexilin, causing cardiomyocyte contractile defects. Intriguingly, we also showed that RBPMS interacts with other cardiac RNA binding proteins, such as RBM20, suggesting their synergistic effects on cardiomyocyte physiology. Our work provides novel insights into how RNA binding proteins regulate cardiomyocyte proliferation and contraction by mediating alternative RNA splicing.
Abstract P1150: Integrative Transcriptomics And Cell Systems Analyses Reveal Protective Pathways Controlled By Igfbp-3 In Anthracycline-induced Cardiotoxicity
Authors: Junjie Chen, Jerome Awada, Mackram Hassanieh, Rene R Packard
Journal: Circulation Research (2023)

Rationale: Anthracyclines such as doxorubicin (Dox) are effective chemotherapeutic agents. Their use however is hindered by the risk of anthracycline-induced cardiotoxicity (AIC). Our understanding of protective pathways activated by cardiomyocytes following AIC remains incomplete. Insulin-like growth factor binding protein (Igfbp)-3, the most abundant Igfbp family member in the circulation, is associated with effects on the metabolism, proliferation, and survival of various cell types. Whereas Igfbp-3 is significantly induced by Dox in the heart, its role in AIC remains ill-defined. Methods and Results: We investigated molecular mechanisms as well as systems-level transcriptomic consequences of manipulating Igfbp-3 in AIC using neonatal rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our findings reveal that Dox induces the nuclear enrichment of Igfbp-3 in cardiomyocytes. Furthermore, Igfbp-3 overexpression leads to a significant reduction of γ-H2AX, a marker of DNA damage (56% reduction vs. control, n=3, P&lt;0.05), and impedes topoisomerase (Top)-2β expression which forms a Top2β-Dox-DNA cleavage complex leading to DNA double-strand breaks (44% reduction vs. control, n=3, P&lt;0.01). Next generation sequencing and gene network analyses of iPSC-CMs treated with Dox reveal that Igfbp-3 perturbation modulates genes implicated in microtubule function and cardiomyocyte contraction. Stimulated Emission Depletion (STED) microscopy and immunoblotting further indicate that Igfbp-3 knockdown exacerbates detyrosinated microtubule accumulation (STED, 21% increase vs. control, n=16-19, P&lt;0.05; immunoblotting, 62% increase vs. control, n=6, P&lt;0.01) – a hallmark of increased cardiomyocyte stiffness and heart failure – and favorably affects contractility following Dox treatment. Conclusion: Our delineation of the protective role of Igfbp-3 in AIC provides novel insight into pathways induced by cardiomyocytes to mitigate DNA damage and apoptosis, and improve contractile function.